Loss of estrogen receptor alpha (ERa) in breast cancer correlates with a more aggressive, tamoxifen resistant phenotype. ERa-negative tumors often display overexpression or amplification of growth factor receptors of the erbB family, particularly EGFR and erbB-2, and consequently, elevated growth factor signaling and resultant MAP kinase (ERK) activity. We have previously shown that overexpression of EGFR or erbB-2, or constitutive activation of Raf or MEK in ERa+, estrogen-dependent MCF-7 cells results in the acquisition of estrogen-independence and loss of ERa expression. We have shown that the common downstream effector of ERa downregulation in all our model cell lines is hyperactive MAPK. Importantly, inhibition of MAPK activity in our cell lines, other ERa- breast cancer cell lines, and breast tumor specimens restores ERa expression. The three specific aims of this proposal are designed to dissect the mechanisms underlying the MAPK induced loss of ERa expression, at both the protein and mRNA levels, and to determine if the reversal of the ERa- phenotype can be extended to clinical breast tumors and results in the in vivo restoration of tamoxifen-sensitivity. They will test the hypotheses that the primary mechanism of downregulation is via proteasomal degradation but transcriptional repression plays an additional, important role in repressing ERa, and that there exists a subpopulation of ERa- tumors in which ERa expression and anti-estrogen response can be restored. Specifically, we will: 1) determine the role of protein degradation, particularly proteasomal degradation mechanisms in MAPK induced repression of ERa expression;2) determine the mechanisms underlying MAPK induced transcriptional repression of ERa;and 3) determine whether the increased ERa mRNA observed upon treatment of tumor specimens correlates with increased ERa protein, the mechanisms underlying lack of restoration in tumors that do not re-express ERa, and using both in vitro and in vivo models, determine if the re-expressed ERa results in restoration of estrogen dependence and anti-estrogen sensitivity. Relevance: The overall goal of this research is to develop novel therapeutic strategies for ER-negative breast. The work proposed here is based on our finding that signaling pathways upregulated in ER- breast cancer cells directly repress ER expression and seeks to determine the mechanisms underlying this in order to identify therapeutic targets that would restore ER expression and anti-estrogen response to ER- breast tumors.